Adipocyte Lipid Research Articles

Usefulness of mean attenuation of pericoronary adipose tissue as a predictor of coronary microvascular dysfunction

Abstract Background/Introduction Coronary microvascular dysfunction (CMD) is a condition in which the distal microvasculature is compromised, resulting in inadequate blood flow to the subsequent arteries and myocardial ischemia. Index of microvascular resistance (IMR), which can quantify CMD, is becoming the standard for the diagnosis of CMD. Patients with CMD have a 4 to 5 times greater risk of cardiovascular death and myocardial infarction than patients without heart disease and similar cardiovascular risk. Early diagnosis of CMD may reduce cardiovascular risk and improve quality of life with therapeutic intervention. Pericoronary adipose tissue mean attenuation (PCATMA) is an indirect measure of adipocyte size and lipid content around coronary artery, reflecting inflammation status. Previous studies have shown that both PCATMA and CMD are associated with the functional severity of coronary artery disease, but no study has yet examined the relationship between PCATMA and CMD. Purpose We conducted a study comparing PCATMA in patients with and without CMD. Methods Between September 2022 and October 2023, 25 patients without significant stenosis (<50% stenosis) who underwent IMR measurement on invasive coronary angiography and coronary CT angiography (CCTA) were included. CMD was diagnosed when the IMR exceeded 25 as measured by a pressure wire inserted into the left anterior descending artery after invasive coronary angiography had excluded significant stenosis. PCATMA was measured on CCTA using the average of the values of right coronary artery, left anterior descending artery, and left circumflex artery. Multivariable logistic regression analysis with stepwise forward selection was used to assess predictors for patients with abnormal IMR. The following variables were included for adjustment: PCATMA, age, sex, diabetes, dyslipidemia, hypertension. Results The mean age of 25 patients in this study was 63 years, 48.0% were female, 29.2% had diabetes, 62.5% had dyslipidemia and 66.7% had hypertension. PCATMA was significantly higher in patients with abnormal IMR (IMR>25) (-79.6 [IQR: -82.1, -74.6] HU vs -86.3 [IQR: -89.8, -78.9] HU, p = 0.043). Multivariate logistic regression analysis demonstrated that PCATMA remained an independent predictor of patients with abnormal IMR (OR 1.27 [95% CI: 1.05–1.65], p = 0.039). Optimal cut-off value of PCATMA for predicting patients with abnormal IMR by Youden index was -82.1 HU (AUC = 0.75 [95% CI: 0.55-0.95]). Conclusions In patients without significant coronary artery stenosis, PCATMA on CCTA may be used to predict the patients with CMD, noninvasively.

Novel injectable polypeptide nanoparticle encapsulated siRNA targeting TGF-β1 and COX-2 for localized fat reduction I: Preclinical invitro and animal models.

Obesity and localized fat accumulation continue to drive the demand for minimally invasive body contouring technologies including injectable compounds for local fat reduction. siRNA offers a potential for an injectable to specifically target and silence genes involved in adipogenesis with minimal inflammatory side effects. This study evaluates the efficacy of STP705, an injectable containing siRNA encapsulated within histidine-lysine polypeptide (HKP) nanoparticles targeting transforming growth factor β1 (TGF-β1) and cyclooxygenase-2 (COX-2), crucial mediators in adipocyte differentiation and fat retention, using invitro, porcine, and murine models. In vitro experiments on mouse preadipocytes and invivo trials using Diet Induced Obese (DIO) mice and Yucatan minipigs were conducted to assess the gene silencing efficiency, tissue localization, pharmacodynamics, and safety profile of STP705. STP705 effectively reduced the expression of TGF-β1 and COX-2, with a notable decrease in adipocyte volume and lipid content without adverse systemic effects. In DIO mice, the HKP-siRNA complex demonstrated precise localization to injected adipose tissue, maintaining significant gene silencing, and detectable levels of siRNA for up to 14 days post-administration. Similar results in minipigs showed a significant reduction in subcutaneous adipose tissue thickness. The results of these studies support the use of targeted siRNA therapy specifically targeting TGF-β1 and COX-2, for localized fat reduction, offering a potential minimally invasive alternative to current fat reduction methods.

Differential chromatin accessibility and Gene Expression Associated with Backfat Deposition in pigs

BackgroundBackfat serves as a vital fat reservoir in pigs, and its excessive accumulation will adversely impact pig growth performance, farming efficiency, and pork quality. The aim of this research is to integrate assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to explore the molecular mechanisms underlying porcine backfat deposition.ResultsATAC-seq analysis identified 568 genes originating from 698 regions exhibiting differential accessibility, which were significantly enriched in pathways pertinent to adipocyte differentiation and lipid metabolism. Besides, a total of 283 transcription factors (TFs) were identified by motif analysis. RNA-seq analysis revealed 978 differentially expressed genes (DEGs), which were enriched in pathways related to energy metabolism, cell cycle and signal transduction. The integration of ATAC-seq and RNA-seq data indicates that DEG expression levels are associated with chromatin accessibility. This comprehensive study highlights the involvement of critical pathways, including the Wnt signaling pathway, Jak-STAT signaling pathway, and fatty acid degradation, in the regulation of backfat deposition. Through rigorous analysis, we identified several candidate genes (LEP, CTBP2, EHHADH, OSMR, TCF7L2, BCL2, FGF1, UCP2, CCND1, TIMP1, and VDR) as potentially significant contributors to backfat deposition. Additionally, we constructed TF-TF and TF-target gene regulatory networks and identified a series of potential TFs related to backfat deposition (FOS, STAT3, SMAD3, and ESR1).ConclusionsThis study represents the first application of ATAC-seq and RNA-seq, affording a novel perspective into the mechanisms underlying backfat deposition and providing invaluable resources for the enhancement of pig breeding programs.

Bioprospection of rattlesnake venom peptide fractions with anti-adipose and anti-insulin resistance activity in vitro

Animal venoms are natural products that have served as a source of novel molecules that have inspired novel drugs for several diseases, including for metabolic diseases such as type-2 diabetes and obesity. From venoms, toxins such as exendin-4 (Heloderma suspectum) and crotamine (Crotalus durissus terrificus) have demonstrated their potential as treatments for obesity. Moreover, other toxins such as Phospholipases A2 and Disintegrins have shown their potential to modulate insulin secretion in vitro. This suggests an unexplored diversity of venom peptides with a potential anti-obesogenic in Mexican rattlesnake venoms. For that reason, this study explored the in vitro effect of Crotalus venom peptide-rich fractions on models for insulin resistance, adipocyte lipid accumulation, antioxidant activity, and inflammation process through nitric oxide production inhibition. Our results demonstrated that the peptide-rich fractions of C. aquilus, C. ravus, and C. scutulatus scutulatus were capable of reverting insulin resistance, enhancing glucose consumption to normal control; C. culminatus, C. molossus oaxacus, and C. polystictus diminished the lipid accumulation on adipocytes by 20%; C. aquilus, C. ravus, and C. s. salvini had the most significant cellular antioxidant activity, having nearly 80% of ROS inhibition. C. aquilus, C. pyrrhus, and C. s. salvini inhibited nitric oxide production by about 85%. We demonstrated the potential of these peptides from Crotalus venoms to develop novel drugs to treat type-2 diabetes and obesity. Moreover, we described for the first time that Crotalus venom peptide fractions have antioxidant and inflammatory properties in vitro models.

Anemarrhena asphodeloides Bunge total saponins lower lipid via modulating MAOA activity to enhance defense mechanisms in mice and C. elegans

Ethnopharmacological relevanceWithin Anemarrhena asphodeloides Bunge (AAB), the pivotal bioactive constituents are identified as Anemarrhena asphodeloides Bunge total saponins (ABS). In traditional pharmacology, ABS has exhibited notable anti-inflammatory, hypoglycemic, and cardioprotective properties. Despite these observed effects, the specific protective mechanisms of ABS against metabolic diseases and improving the endocrine system remain largely uncharted. Aim to studyThis work intends to shed light on the effects and intrinsic mechanisms of ABS on metabolic diseases. Materials and methodsThe characterization of ABS components was achieved through High-Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS). To evaluate ABS's anti-inflammatory efficacy, mouse macrophages underwent analysis using the Griess method. Induced differentiation of mouse fibroblasts was assessed through Oil Red O staining. In an obesity model with C57BL/6 N mice, ABS administration prompted measurements of glucose and insulin tolerance. Western blot analysis quantified lipolysis and anti-inflammatory protein expression. Nile red staining gauged body fat content in C. elegans post-ABS treatment. The mechanism of ABS action was elucidated through mRNA sequencing, further validated using RNA interference technology, and nematode mutants. ResultsABS showcased the ability to diminish Nitric Oxide (NO) production in inflammatory macrophages and shrink adipocyte lipid droplets. In mice experiments, ABS was effective in alleviating fat accumulation and affecting serum lipid metabolism in diabetic mice. It enhanced oral glucose tolerance and insulin tolerance while increasing lipolysis-associated protein expression. ABS notably reduced fat content in C. elegans. Mechanistically, ABS downregulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and monoamine oxidase A (MAOA) expression while enhancing UGT, ilys-2, and ilys-3. Lipolysis emerged as a pivotal pathway for ABS in the therapeutic intervention of metabolic diseases. ConclusionsOur investigation has revealed that ABS exert a role in combating metabolic diseases by enhancing the body's defense mechanisms. ABS activate the NLRP3-neurotransmitter-visceral adipose pathway in mice, thereby bolstering resistance and diminishing fat accumulation. In C. elegans, ABS downregulated the expression of MAOA, bolstered resistance, and augmented glucuronidase activity, consequently leading to a reduction in fat content.

Lycium chinense Mill Induces Anti-Obesity and Anti-Diabetic Effects In Vitro and In Vivo.

This study investigated the effects of Lycium chinense Mill (LCM) extract on obesity and diabetes, using both in vitro and high-fat diet (HFD)-induced obesity mouse models. We found that LCM notably enhanced glucagon-like peptide-1 (GLP-1) secretion in NCI-h716 cells from 411.4 ± 10.75 pg/mL to 411.4 ± 10.75 pg/mL compared to NT (78.0 ± 0.67 pg/mL) without causing cytotoxicity, implying the involvement of Protein Kinase A C (PKA C) and AMP-activated protein kinase (AMPK) in its action mechanism. LCM also decreased lipid droplets and lowered the expression of adipogenic and lipogenic indicators, such as Fatty Acid Synthase (FAS), Fatty Acid-Binding Protein 4 (FABP4), and Sterol Regulatory Element-Binding Protein 1c (SREBP1c), indicating the suppression of adipocyte differentiation and lipid accumulation. LCM administration to HFD mice resulted in significant weight loss (41.5 ± 3.3 g) compared to the HFD group (45.1 ± 1.8 g). In addition, improved glucose tolerance and serum lipid profiles demonstrated the ability to counteract obesity-related metabolic issues. Additionally, LCM exhibited hepatoprotective properties by reducing hepatic lipid accumulation and diminishing white adipose tissue mass and adipocyte size, thereby demonstrating its effectiveness against hepatic steatosis and adipocyte hypertrophy. These findings show that LCM can be efficiently used as a natural material to treat obesity and diabetes, providing a new approach for remedial and therapeutic purposes.

The Inhibitory Effects of Maclurin on Fatty Acid Synthase and Adipocyte Differentiation.

Obesity is a complex health condition characterized by excessive adipose tissue accumulation, leading to significant metabolic disturbances such as insulin resistance and cardiovascular diseases. Fatty acid synthase (FAS), a key enzyme in lipogenesis, has been identified as a potential therapeutic target for obesity due to its role in adipocyte differentiation and lipid accumulation. This study employed a multidisciplinary approach involving in silico and in vitro analyses to investigate the anti-adipogenic properties of maclurin, a natural phenolic compound derived from Morus alba. Using SwissDock software (ChEMBL version 23), we predicted protein interactions and demonstrated a high probability (95.6%) of maclurin targeting FAS, surpassing the interaction rates of established inhibitors like cerulenin. Docking simulations revealed maclurin's superior binding affinity to FAS, with a binding score of -7.3 kcal/mol compared to -6.7 kcal/mol for cerulenin. Subsequent in vitro assays confirmed these findings, with maclurin effectively inhibiting FAS activity in a concentration-dependent manner in 3T3-L1 adipocytes, without compromising cell viability. Furthermore, maclurin treatment resulted in significant reductions in lipid accumulation and the downregulated expression of critical adipogenic genes such as PPARγ, C/EBPα, and FAS, indicating the suppression of adipocyte differentiation. Maclurin shows potential as a novel FAS inhibitor with significant anti-adipogenic effects, offering a promising therapeutic avenue for the treatment and prevention of obesity.

Navigating Lipodystrophy: Insights from Laminopathies and Beyond.

Recent research into laminopathic lipodystrophies-rare genetic disorders caused by mutations in the LMNA gene-has greatly expanded our knowledge of their complex pathology and metabolic implications. These disorders, including Hutchinson-Gilford progeria syndrome (HGPS), Mandibuloacral Dysplasia (MAD), and Familial Partial Lipodystrophy (FPLD), serve as crucial models for studying accelerated aging and metabolic dysfunction, enhancing our understanding of the cellular and molecular mechanisms involved. Research on laminopathies has highlighted how LMNA mutations disrupt adipose tissue function and metabolic regulation, leading to altered fat distribution and metabolic pathway dysfunctions. Such insights improve our understanding of the pathophysiological interactions between genetic anomalies and metabolic processes. This review merges current knowledge on the phenotypic classifications of these diseases and their associated metabolic complications, such as insulin resistance, hypertriglyceridemia, hepatic steatosis, and metabolic syndrome, all of which elevate the risk of cardiovascular disease, stroke, and diabetes. Additionally, a range of published therapeutic strategies, including gene editing, antisense oligonucleotides, and novel pharmacological interventions aimed at addressing defective adipocyte differentiation and lipid metabolism, will be explored. These therapies target the core dysfunctional lamin A protein, aiming to mitigate symptoms and provide a foundation for addressing similar metabolic and genetic disorders.

Dahuang Huanglian Xiexin Decoction ameliorates obesity via modulating adipocyte differentiation and lipid degradation through inhibiting endoplasmic reticulum stress

Dahuang Huanglian Xiexin Decoction (DHXD) is the representative clinical formula for treating epigastric oppression. In this study, we aim to explore the effect of DHXD on obesity and attempt to investigate its potential mechanism. 3T3-L1 preadipocytes were differentiated and high-fat diet-induced obese rat model was established. DHXD was used for treatment and tunicamycin, the activator of endoplasmic reticulum (ER) stress, was adopted to investigate the related regulatory mechanism. Cell viability was evaluated using CCK-8 assay. Oil-Red O staining was performed to determine lipid accumulation. Glycerol production and Triglyceride content were measured using their commercial kits. Western blot was conducted to examine the expression of critical proteins. Results indicated that DHXD could greatly reduce intracellular lipid droplets and triglyceride in differentiated 3T3-L1 cells. Moreover, the elevated expression of mature adipocytes markers, PPARγ, aP2, during adipogenesis was decreased by DHXD treatment. In addition, DHXD aggravated the lipolysis in differentiated 3T3-L1 cells, as evidenced by the upregulated ATGL expression and the downregulated HSL expression. Besides, DHXD inhibited endoplasmic reticulum (ER) stress in 3T3-L1 cells. Further experiments indicated that the impacts of DHXD on adipocyte differentiation and lipid degradation were partly abolished by tunicamycin. Finally, DHXD alleviated lipid accumulation and ER stress in obese rats. In conclusion, DHXD ameliorates obesity via modulating adipocyte differentiation and lipid degradation through inhibiting ER stress.

Downregulation of adipose LPL by PAR2 contributes to the development of hypertriglyceridemia.

Lipoprotein lipase (LPL) hydrolyzes circulating triglycerides (TGs), releasing fatty acids (FA) and promoting lipid storage in white adipose tissue (WAT). However, the mechanisms regulating adipose LPL and its relationship with the development of hypertriglyceridemia are largely unknown. WAT from obese humans exhibited high PAR2 expression, which was inversely correlated with the LPL gene. Decreased LPL expression was also inversely correlated with elevated plasma TG levels, suggesting that adipose PAR2 might regulate hypertriglyceridemia by downregulating LPL. In mice, aging and high palmitic acid diet (PD) increased PAR2 expression in WAT, which was associated with a high level of macrophage migration inhibitory factor (MIF). MIF downregulated LPL expression and activity in adipocytes by binding with CXCR2/4 receptors and inhibiting Akt phosphorylation. In a MIF overexpression model, high-circulating MIF levels suppressed adipose LPL, and this suppression was associated with increased plasma TGs but not FA. Following PD feeding, adipose LPL expression and activity were significantly reduced, and this reduction was reversed in Par2-/- mice. Recombinant MIF infusion restored high plasma MIF levels in Par2-/- mice, and the levels decreased LPL and attenuated adipocyte lipid storage, leading to hypertriglyceridemia. These data collectively suggest that downregulation of adipose LPL by PAR2/MIF may contribute to the development of hypertriglyceridemia.

Extracellular matrix modulates depot-specific adipogenic capacity in adipose tissue of dairy cattle

Adipose tissue (AT) expands through both hyperplasia and hypertrophy. During adipogenesis, adipose stromal and progenitor cells (ASPCs) proliferate and then accumulate lipids, influenced by the local AT microenvironment. Increased adipogenic capacity is desirable as it relates to metabolic health, especially in transition dairy cows where excess free fatty acids in circulation can compromise metabolic and immune health. Our aim was to elucidate the depot-specific adipogenic capacity and ECM properties of subcutaneous (SAT) and visceral (VAT) AT of dairy cows and define how the ECM affects adipogenesis. Flank SAT and omental VAT samples were collected from dairy cows in a local abattoir. Tissue samples were utilized for transcriptome analysis, targeted RT-qPCR for adipogenic markers, adipocyte sizing, assessment of viscoelastic properties and collagen accumulation, and then decellularized for native ECM isolation. For in vitro analyses, SAT and VAT samples were digested via collagenase, and ASPCs cultured for metabolic analysis. Adipogenic capacity was assessed by adipocyte size, quantification of ASPCs in stromal vascular fraction (SVF) via flow cytometry, and gene expression of adipogenic markers. In addition, functional assays including lipolysis and glucose uptake were performed to further characterize SAT and VAT adipocyte metabolic function. Data were analyzed using SAS (version 9.4; SAS institute Inc., Cary, NC) and GraphPad Prism 9. Subcutaneous AT adipogenic capacity was greater than VAT's, as indicated by increased ASPCs abundance, increased magnitude of adipocyte ADIPOQ and FASN expression during differentiation, and higher adipocyte lipid accumulation as shown by an increased proportion of larger adipocytes and abundance of lipid droplets. Rheologic analysis revealed that VAT is stiffer than SAT, which led us to hypothesize that differences between SAT and VAT adipogenic capacity were partly mediated by depot-specific ECM microenvironment. Thus, we studied depot-specific ECM-adipocyte crosstalk using a 3D model with native ECM (decellularized AT). Subcutaneous AT and VAT ASPCs were cultured and differentiated into adipocytes within depot-matched and mis-matched ECM for 14d, followed by ADIPOQ expression analysis. Visceral AT ECM impaired ADIPOQ expression in SAT cells. Our results demonstrate that SAT is more adipogenic than VAT and suggest that divergences between SAT and VAT adipogenesis are partially mediated by the depot-specific ECM microenvironment.

Red Oranges and Olive Leaf Waste-Derived Bioactive Extracts Promote Adipocyte Functionality In Vitro.

Obesity is increasingly prevalent worldwide and is linked to metabolic diseases, such as insulin resistance (IR) and type 2 diabetes mellitus (T2DM), due to excessive free fatty acids (FFAs). Although lifestyle changes are effective, they often prove to be insufficient as initial treatments for obesity. Additionally, while surgical and pharmacological interventions are available, they are not entirely safe or effective. Recently, interest has grown in utilizing food waste and plant-derived phenolic compounds for their health benefits, presenting a promising avenue for managing obesity and its related disorders. Indeed, many studies have examined the potential inhibitory effects of the natural extract on adipocyte differentiation and lipid accumulation. This study focused on the evaluation of the effects of standardized extracts obtained from red oranges and olive leaf waste on 3T3-L1 murine pre-adipocyte and adipocyte functionality. Red orange extract (ROE) and olive leaf extract (OLE), alone and in combination, were tested to assess their anti-obesity and anti-inflammatory effects, as well as their potential therapeutic benefits. Three in vitro models were established to investigate the effects of the extracts on (I) adipocyte differentiation; (II) mature and hypertrophic adipocytes challenged with palmitic acid (PA) and erastin (ER), respectively; and (III) erastin-induced cytotoxicity on pre-adipocytes.

Nkx1.2 deletion decreases fat production in zebrafish.

This study aimed to investigate the role of Nkx1-2, a transcription factor with the NK homeobox domain, in the regulation of fat production. Gene expression was analyzed using quantitative real-time polymerase chain reaction or transcriptome sequencing. CRISPR/Cas9 technology was employed to generate nkx1.2 knockout zebrafish and nkx1.2-deleted 3T3-L1 cells. Lipid droplet production in zebrafish larvae was visually quantified using Nile red staining, whereas lipid droplets in 3T3-L1 cells were stained with Oil red O. The binding of Nkx1-2 to the promoter was verified through an electrophoretic mobility shift assay experiment. Nkx1-2 plays crucial roles in the regulation of fat production in zebrafish. Knockout of nkx1.2 in zebrafish leads to weight loss, accompanied by significantly reduced lipid droplet production and decreased visceral and liver fat content. Furthermore, genes related to lipid biosynthesis are significantly downregulated. In 3T3-L1 preadipocytes, Nkx1-2 induces differentiation into mature adipocytes by binding to the cebpa promoter, thereby activating its transcription. Additionally, the expression of nkx1.2 is regulated by the p38 MAPK, JNK, or Smad2/3 signaling pathways in 3T3-L1 cells. Our findings suggest that Nkx1-2 functions as a positive regulator of fat production, playing a critical role in adipocyte differentiation and lipid biosynthesis.

GRK5 is required for adipocyte differentiation through ERK activation.

Previous studies have identified G protein-coupled receptor (GPCR) kinase 5 (GRK5) as a genetic factor contributing to obesity pathogenesis, but the underlying mechanism remains unclear. We demonstrate here that Grk5 mRNA is more abundant in stromal vascular fractions of mouse white adipose tissue, the fraction that contains adipose progenitor cells, or committed pre-adipocytes, than in adipocyte fractions. Thus, we generated a GRK5 knockout (KO) 3T3-L1 pre-adipocyte to further investigate the mechanistic role of GRK5 in regulating adipocyte differentiation. During adipogenic stimulation, GRK5 KO pre-adipocytes were unable to achieve mature adipocyte morphology and lipid accumulation compared to wildtype cells coupled with suppressed adipogenic and lipogenic gene expression. Beside GPCR signaling, RNA sequencing and pathway analysis identified insulin-like growth factor 1 (IGF-1) signaling to be one of the top 5 significantly dysregulated pathways in GRK5 KO cells. GRK5 KO cells also displayed decreased insulin-stimulated ERK phosphorylation, a downstream target of insulin-stimulated IGF-1 receptor activation, suggesting that GRK5 acts through this critical pathway to impact 3T3-L1 adipocyte differentiation. To find a more translational approach, we identified a new small molecule GRK5 inhibitor that was able to reduce 3T3-L1 adipogenesis. These data suggest that GRK5 is required for adipocyte differentiation through IGF-1 receptor/ERK activation and may be a promising translational target for obesity.

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

Abstract 125: The X Chromosome Gene Kdm6a Confers Sex Differences In Mature Adipocyte Function Impacting Cardiometabolic Health

Biological sex impacts cardiovascular disease and other metabolic conditions that increase disease risk, including obesity. Physiological sex differences are determined by two components of sex: gonadal hormones and sex chromosome complement. Using mouse models to dissociate gonadal sex from chromosomal sex, we have identified sex chromosome-dependent regulation of circulating lipoproteins, atherosclerosis, and adiposity. Specifically for adiposity, mice with two X chromosomes had higher body weight gain compared to mice with XY sex chromosomes in the presence of ovaries or testes. In cells with XX chromosome complement, specific X chromosome genes escape X inactivation, which leads to higher expression in XX (female) compared to XY (male) tissues. We identified an X escape gene, Kdm6a , that encodes a histone demethylase enzyme and impacts adiposity by altering the function of mature adipocytes. Mice with mature adipocyte-specific Kdm6a reduction (Ad- Kdm6a -/- ) had lower body weight and adiposity compared to wildtype females. The white adipose triglyceride pool was altered with higher unsaturated and long chain fatty acids in Ad- Kdm6a -/- mice compared to wildtype mice. Furthermore, siRNA knockdown of Kdm6a in cultured adipocytes altered lipid metabolism with reduced lipid droplet size. Given that KDM6A is a histone demethylase, we assessed the Kdm6a dosage effect on adipocyte gene expression and histone modifications across the genome. Differential gene expression associated with lipid homeostasis was identified in Ad- Kdm6a -/- mice and in cultured adipocytes with Kdm6a knockdown compared to controls. KDM6A genomic targets and resultant histone modifications were identified in cultured adipocytes using CUT&RUN. We conclude that differential gene dosage of Kdm6a between female and male tissues is a determinant of adipose sex differences through modulation of histone modifications and gene expression. This leads to effects on adipocyte lipid composition and tissue mass, which impacts susceptibility to obesity and metabolic disease.

MARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Smooth muscle cell-derived Cxcl12 directs macrophage accrual and sympathetic innervation to control thermogenic adipose tissue

Sympathetic innervation of brown adipose tissue (BAT) controls mammalian adaptative thermogenesis. However, the cellular and molecular underpinnings contributing to BAT innervation remain poorly defined. Here, we show that smooth muscle cells (SMCs) support BAT growth, lipid utilization, and thermogenic plasticity. Moreover, we find that BAT SMCs express and control the bioavailability of Cxcl12. SMC deletion of Cxcl12 fosters brown adipocyte lipid accumulation, reduces energy expenditure, and increases susceptibility to diet-induced metabolic dysfunction. Mechanistically, we find that Cxcl12 stimulates CD301+ macrophage recruitment and supports sympathetic neuronal maintenance. Administering recombinant Cxcl12 to obese mice or leptin-deficient (Ob/Ob) mice is sufficient to boost macrophage presence and drive sympathetic innervation to restore BAT morphology and thermogenic responses. Altogether, our data reveal an SMC chemokine-dependent pathway linking immunological infiltration and sympathetic innervation as a rheostat for BAT maintenance and thermogenesis.

Effects of a Global Rab27a Null Mutation on Murine PVAT and Cardiovascular Function.

RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.

Mechanistic insights into the role of USP14 in adipose tissue macrophage recruitment and insulin resistance in obesity

Diet-induced obesity can cause metabolic syndromes. The critical link in disease progression is adipose tissue macrophage (ATM) recruitment, which drives low-level inflammation, triggering adipocyte dysfunction. It is unclear whether ubiquitin-specific proteinase 14 (USP14) affects metabolic disorders by mediating adipose tissue inflammation. In the present study, we showed that USP14 is highly expressed in ATMs of obese human patients and diet-induced obese mice. Mouse USP14 overexpression aggravated obesity-related insulin resistance by increasing the levels of pro-inflammatory ATMs, leading to adipose tissue inflammation, excessive lipid accumulation, and hepatic steatosis. In contrast, USP14 knockdown in adipose tissues alleviated the phenotypes induced by a high-fat diet. Co-culture experiments showed that USP14 deficiency in macrophages led to decreased adipocyte lipid deposition and enhanced insulin sensitivity, suggesting that USP14 plays an important role in ATMs. Mechanistically, USP14 interacted with TNF receptor-associated 6, preventing K48-linked ubiquitination as well as proteasome degradation, leading to increased pro-inflammatory polarization of macrophages. In contrast, the pharmacological inhibition of USP14 significantly ameliorated diet-induced hyperlipidemia and insulin resistance in mice. Our results demonstrated that macrophage USP14 restriction constitutes a key constraint on the pro-inflammatory M1 phenotype, thereby inhibiting obesity-related metabolic diseases.